Members of the Smad family of Transforming Growth Factor-beta (TGF-beta) signal transducers act as tumor suppressors in a variety of tissues. Homozygous mutations in Smad2 or Smad4 are detected in 50% of pancreas, 20% of colon and 10% of lung tumors. However, the TGF-beta signals and target genes that normally suppress proliferation in these tissues are unknown. As a result, the mechanism by which Smad mutations result in tumors is also unknown. In the absence of this information, the current hypothesis is that all Smad tumor mutations are loss of function mutations and that they all lead to tumors via a single mechanism. In Specific Aim 1 we test the first part of the hypothesis. We will determine if any Smad tumor alleles are gain of function mutations. To do this we will exploit a well- established feature of the Smad family, the ability of homologous family members to function across species. We will express Smad mutant alleles isolated from human tumors in Drosophila. We will compare phenotypes generated by the Smad mutant alleles to the phenotypes generated by Smad wild type alleles we have recently published. Our preliminary studies show that the first part of the hypothesis is false. We have identified a gain of function mutation in Smad4. In Specific Aim 2 we will test the second part of the hypothesis. We will conduct ?mechanism of action? screens to identify genes involved in Smad gain of function mutant phenotypes. We will isolate mutations that enhance or suppress the gain of function phenotype and then clone the responsible gene. The rationale is that Drosophila genes that enhance Smad gain of function phenotypes likely have human homologs that assist Smad mutants in tumorigenesis and genes that suppress these phenotypes have homologs that antagonize tumor formation. Screens involving different phenotypes may identify distinct sets of genes suggesting that Smad mutants induce tumors by multiple mechanisms. This study will provide unprecedented insight into Smad mutant alleles and the mechanisms they use to generate tumors. This knowledge can be used to determine if Smad proteins should be exploited as anti-cancer drug discovery targets.